Therapeutic composition with a botulinum neurotoxin

ABSTRACT

The present invention pertains to pharmaceutical compositions which comprise a botulinum neurotoxin from  Clostridium botulinum , the neurotoxin being free of the complexing proteins naturally present in the botulinum neurotoxin complex or being chemically modified or being modified by genetic manipulation. Moreover the pharmaceutical compositions of the instant invention have good stability and are advantageously formulated free of human serum albumin.

The present invention pertains to pharmaceutical compositions whichcomprise a botulinum neurotoxin from Clostridium botulinum , theneurotoxin being free of the complexing proteins naturally present inthe botulinum neurotoxin complex, or a botulinum neurotoxin which ismodified chemically or modified by genetic manipulation said modifiedbotulinum toxin being free of the complexing proteins which naturallyform complexes with botulinum neurotoxins. Moreover the pharmaceuticalcompositions of the instant invention have good stability and areadvantageously formulated free of human serum albumin.

Albumin of human origin has been utilized as a bulk carrier andstabilizer for protein active ingredients present in pharmaceuticalcompositions. Albumin has been demonstrated to stabilize proteinaceousactive ingredients in pharmaceutical compositions by reducing adhesionand reducing denaturation of the active ingredient. Moreover, thealbumin has no immunogenicity on injection into a human patient.

Significant drawbacks exist, however, to the use of albumin in apharmaceutical composition. Albumin has been attributed to transmissionof certain stable viruses, prions or other infectious or pathogeniccompounds, e.g., human transmissible spongiform encephalopathy (TSE).Consequently, there is increased regulatory scrutiny of pharmaceuticalcompositions containing human serum albumin. Similarly, gelatin has beenused in some pharmaceutical compositions containing protein activeingredients as an albumin substitute. As a mammalian derived protein,gelatin also poses the same risk of pathogen transmission. Therefore,there is a need for a replacement for mammalian derived proteinaceousstabilizers.

Botulinum toxin complexes are composed of a mixture of clostridialproteins. These are hemagglutinins with different molecular masses, anontoxic, non-hemagglutinating protein (Mr about 120,000) and aneurotoxin (Mr about 150,000). They form an acid-stable complex which isresponsible for the oral toxicity in cases of food poisoning. Incontrast to the pure neurotoxin, the complex resists the aggressiveenvironment in the gastrointestinal tract and makes enteral absorptionof the neurotoxin possible, and this reaches the target cells via thebloodstream or the lymphatic system and there induces blockade ofneurotransmitter release. This is followed by muscle paralysis andcessation of various autonomic functions. Poisoned patients die ofrespiratory muscle failure. Since the pure neurotoxin is degraded in thegastrointestinal tract and thus does not undergo enteral absorption, itis not toxic after ingestion. On parenteral administration, thetherapeutic effects of the neurotoxin and of the complex do not differsince the complex decomposes into its constituents in tissue, and onlythe neurotoxin is taken up by the target cells.

At present two products comprising botulinum neurotoxin type A areapproved for the treatment of blepharospasm, hemifacial spasms andspasmodic torticollis: BOTOX® and DYSPORT®. The botulinum neurotoxin isin the current state of the art injected directly into dystonic orspastic muscles, where the neurotoxin is released at physiological pHfrom the complex and elicits the desired pharmacological effect.Clinical trials for treating other disorders of the nervous system (e.g.spasticities, migraine, low back pain, cervical spine disorders,hypersalivation) are currently in progress. Botulinum toxin complex typeA (Mr 900,000) is approved for the therapy of various dystonias. Theapproved products are also employed for cosmetic indications such ashyperhidrosis and pronounced wrinkling. The other Clostridium botulinumtoxin complexes (of types B, C1, D, E, F, G) as well as toxins which arederived from these Clostridium botulinum toxins by chemical modificationor genetic manipulation are also suitable for these therapies.

Both BOTOX® and DYSPORT® are provided to clinicians in lyophilized formfor reconstitution just prior to use. Unfortunately, not every patientand indication requires the same dosage. Consequently, reconstitutedcomposition is often either frozen or refrigerated for later use. Theseheld over reconstituted compositions have been evaluated for potencystability. It has been observed that BOTOX® looses at least 44% of itspotency when it is reconstituted and stored under refrigeration for 12hours. Moreover, when the reconstituted composition is frozen at −70°C., it loses 70% of its potency. Gartlan, M. G., and Hoffman, H. T.Crystalline preparation of botulinum toxin type A (Botox): Degradationin potency with storage., Otolaryngology—Head and Neck Surgery 102 (2):135-140 (1992). Such instability results in significant dosage variationand wasted product. Thus, an object of the instant invention is thedevelopment and production of a stable liquid and freeze-driedformulation of botulinum toxin which has better handling characteristicsthan existing formulations.

A novel pharmaceutical composition has been developed which comprisesbotulinum neurotoxin (type A, B, C1, D, E, F, or G) which is free ofhemagglutinins and other exogenous proteins. This reduces the totalprotein load of a pharmaceutical formulation without reduction of theamount of toxin. We found in antigenicity studies that the pureneurotoxin of all types, in contrast to commercial products of type Aand the complexes of types B to G, induces no, or at the most verylittle, formation of antibodies. On therapeutic use of this newlydeveloped pharmaceutical (pure neurotoxin of types A, B, C1, D, E, F, orG), there is no failure of therapy due to antibodies even after repeatedadministration.

However, and as noted above, problems arise on formulation. As aproteinaceous active, botulinum toxin is very labile. Furthermore,botulinum toxin complexes are extremely susceptible to denaturation dueto surface denaturation, heat, and alkaline conditions.

As with enzymes generally, the biological activities of botulinum toxins(which are intracellular peptidases) is dependant, at least in part,upon their three dimensional conformation. Thus, botulinum toxins may bedetoxified by heat, various chemicals, surface stretching and surfacedrying. Additionally, it is known that the dilute toxin concentrationsused in the approved indications result in rapid detoxification of thetoxin unless a suitable stabilizing agent is present. Moreover, toxinstability is a significant factor on storage. Consequently, stabilizingagents are essential. To date, stability has been achieved throughformulation in mammalian derived proteins albumin and gelatin. As notedabove, mammalian derived proteins raise the risk that certain stableviruses, prions or other infectious or pathogenic compounds carriedthrough from donors may contaminate the toxin.

Furthermore, the conditions on lyophilization, including pH,temperature, dilution and vacuum pressures operate to detoxify thetoxin. To date, mammalian derived proteins such as gelatin and serumalbumin have been used with some success to stabilize botulinum toxinand are the standard stabilizers.

For example, the commercially available botulinum toxin containingpharmaceutical composition BOTOX® (available from Allergan, Inc., ofIrvine, Calif.) consists of a purified botulinum toxin type A complex,albumin and sodium chloride packaged in sterile, vacuum-dried form. Eachvial of BOTOX® contains about 100 units (U) of Clostridium botulinumtoxin type A complex, 0.5 milligrams of human serum albumin and 0.9milligrams of sodium chloride in a sterile, vacuum-dried form without apreservative.

The art is replete with attempts to stabilize protein compositions.Carpender et al., Interactions of Stabilizing Additives with ProteinsDuring Freeze-Thawing and Freeze-Drying, International Symposium onBiological Product Freeze-Drying and Formulation, 24-26 Oct. 1990;Karger (1992), 225-239.

The use of the disaccharide cellobiose as an excipient in conjunctionwith albumin and sodium chloride demonstrates toxicity degradation (10%recovery) after lyophilization of crystalline botulinum toxin type Awith these excipients, as compared to the toxicity after lyophilizationwith only albumin (>75% to >90% recovery). Goodnough et al.,Stabilization of Botulinum Toxin Type A During Lyophilization, App &Envir. Micro. 58 (10) 3426-3428 (1992).

Moreover, protein formulations comprising a saccharide (such as glucoseor a polymer of glucose) or carbohydrates are not known to be stablebecause proteins and glucose have been demonstrated to interact togetherand to undergo Maillard degradation, due to the reducing nature ofglucose and glucose polymers. In contrast, alcohols, e.g., inositol,mannitol, are non-reducing and have long been employed as cryoprotectantexcipients to stabilize proteins during lyophilization.

In light of the instability of botulinum toxin and the attendant risksof mammalian derived stabilizers and polysaccharides, a suitable proteinstabilizer continues to be an objective for formulation scientists.

SUMMARY

It is an object of the present invention to provide a non-proteinaceousreplacement for mammalian derived proteins in pharmaceuticalcompositions containing botulinum toxin or a toxin which is derived frombotulinum toxin by chemical modification or by genetic manipulation.This new formulation must have low, and preferably negligible,immunogenicity when injected into a human patient.

DEFINITIONS

As used herein, the words or terms set forth below have the followingdefinitions.

“Pharmaceutical composition” is a formulation in which an activeingredient, in this case a botulinum toxin, or the instant hemagglutininprotein free botulinum toxin, or a toxin which is derived from botulinumtoxin by chemical modification or by genetic manipulation, is stabilizedby a substance other than a mammalian derived protein. Suchpharmaceutical composition may be suitable for diagnostic or therapeuticadministration (i.e. by intramuscular or subcutaneous injection) to ahuman patient. The pharmaceutical composition may be lyophilized orvacuum dried, reconstituted, or in solution. The botulinum toxin activeingredient may be one of the botulinum toxin serotypes A, B, C1, D, E, For G, all of which may be further modified to be free of the complexingproteins present in natural neurotoxin or modified chemically ormodified by genetic manipulation.

“Therapeutic formulation” refers to the capacity of the instantformulation to treat/alleviate a condition such as a condition incidentto hyperactivity (i.e. spasticity) of a peripheral muscle.

“Stabilizing”, stabilizes” or “stabilization” means that the activeingredient, i.e., a botulinum toxin or a toxin which is derived frombotulinum toxin by chemical modification or by genetic manipulationpresent in a reconstituted or aqueous solution pharmaceuticalcomposition has greater than about 20% and up to about 100% of thetoxicity that the biologically active botulinum toxin had prior to beingincorporated into the pharmaceutical composition.

“Cryoprotectant” refers to excipients which result in the activeingredient, i.e., a botulinum toxin or a toxin which is derived frombotulinum toxin by chemical modification or by genetic manipulationpresent in a reconstituted or aqueous solution pharmaceuticalcomposition having greater than about 20% and up to about 100% of thetoxicity that the biologically active botulinum toxin had prior to beingfreeze-dried in the pharmaceutical composition.

“pH buffer” refers to chemical substances being capable to adjust the pHvalue of a composition, solution and the like to a certain value or to acertain pH range.

“Polyalcohol” refers to aliphatic or cycloaliphatic carbohydratesbearing more than one hydroxyl functional group but no carbonylfunctional group (like, for instance, in sugar compounds).

“Free from mammalian derived protein stabilizing agents” means that thecomposition or preparation does not contain detectable amounts ofstabilizing agents derived from mammalian proteins.

“Chemical modification” refers to methods known in the art for modifyingthe native botulinum toxin of any serotype by means of chemicalreactions or the like; it refers especially to substitutions, deletions,insertions, additions or posttranslational modifications of amino acidsof the botulinum toxin.

“Genetic manipulation” refers to methods known in the art for modifyingthe native botulinum toxin of any serotype by means of modifying thegene encoding for the botulinum toxin or respective nucleic acids likeDNA or RNA.

DESCRIPTION

The present invention describes the discovery that a stable neurotoxincontaining pharmaceutical composition may be formulated free of anymammalian derived protein or donor pool albumin by incorporating anon-proteinaceous stabilizing agent, especially by incorporatinghyaluronic acid or polyvinylpyrrolidone or polyethyleneglycol or amixture of two or more thereof. The instant invention pertains to thedevelopment of a botulinum toxin composition which is formulated with ahyaluronic acid or polyvinylpyrrolidone or polyethyleneglycol or amixture of two or more thereof. Such composition is a safer compositionpossessing remarkable stability.

Fortunately, the instant composition is significant in that thebotulinum toxin or a toxin which is derived from botulinum toxin bychemical modification or by genetic manipulation is not formulated in amammalian derived proteinaceous stabilizer. It has been determined thata hyaluronic acid or polyvinylpyrrolidone or polyethyleneglycolformulation or mixtures thereof, particularly those incorporating a pHbuffer, especially sodium acetate buffer, and/or a cryoprotectant, mayfunction to increase the stability and useful storage life of theinstant pharmaceutical composition.

Furthermore, the instant pharmaceutical composition or preparationpreferably is not only free of a mammalian derived proteinaceousstabilizing agent but free of any stabilizing protein.

The instant invention is not limited to pharmaceutical compositions, butalso refers to processes for stabilizing botulinum toxin compositions orcompositions of a toxin which is derived from botulinum toxin bychemical modification or by genetic manipulation. By incorporatinghyaluronic acid or polyvinylpyrrolidone or polyethyleneglycol ormixtures thereof into the composition, the botulinum toxin or the toxinwhich is derived from botulinum toxin by chemical modification or bygenetic manipulation is stabilized. Moreover, stability may be enhancedby incorporating a pH buffer into the pharmaceutical composition,thereby stabilizing pH and contributing to reconstituted toxin shelflife and/or by including a cryoprotectant into the pharmaceuticalcomposition, thereby increasing freeze-dry stability and shelf life.

It is preferred that the pH buffer in its nature and amount is capableof stabilizing or adjusting the pH of the instant composition orpreparation to values in the range of approximately 4 to about 7.5.Suitable pH buffers may be citrate, phosphate and especially acetatebuffer systems, in particular sodium acetate buffer systems.Surprisingly, it has been found that if an acetate buffer is used in theinstant composition or preparation and said composition or preparationis freeze-dried, the acetate may be removed from the composition orpreparation during freeze-drying; after reconstituting or thawing thecomposition or preparation has an approximately neutral pH (in the rangeof about 6.5 to about 7.5). This is advantageous when the composition orpreparation is administered to a person or patient in need thereof,especially when injected into a muscle, because the then (almost)neutral botulinum toxin composition or preparation causes less pain thana respective composition or preparation having an acidic pH of, forinstance, 4.

A detailed embodiment of the present invention may be a pharmaceuticalcomposition suitable for injection into a human patient, which includesa botulinum toxin or a toxin which is derived from botulinum toxin bychemical modification or by genetic manipulation, and a hyaluronic acidor a polyvinylpyrrolidone or a polyethleneglycol, such composition beingoptionally pH stabilized by a suitable pH buffer, in particular by asodium acetate buffer, and/or a cryoprotectant polyalcohol.

The pharmaceutical composition is suitable for administration to a humanpatient to achieve a therapeutic effect, and the neurotoxin may be oneof the botulinum toxin serotypes A, B, C1, D, E, F and G, preferably abotulinum toxin which is free of the complexing proteins present innatural neurotoxin or neurotoxin modified chemically or modified bygenetic manipulation. The modified neurotoxin is free of the complexingproteins which naturally form complexes with botulinum neurotoxin aswell.

The modification of the neurotoxin derived from botulinum neurotoxin dueto chemical modifying or genetic manipulation can be located on eachpart of the neurotoxin protein, for instance on the heavy chain partand/or on the light chain part of the neurotoxin molecule. There mightbe one modification or more modifications. Preferably, the heavy chainof the neurotoxin protein derived from botulinum neurotoxin comprisesone or more modifications which may decrease or increase the affinity ofthe neurotoxin for binding to nerve cells when compared to the nativeneurotoxin. Such modified neurotoxin may comprise at least onesubstitution and/or deletion and/or insertion and/or addition and orposttranslational modification of amino acids of the neurotoxin andpreferably of the heavy chain of the neurotoxin.

Whether the pharmaceutical composition comprises, beside the neurotoxinactive ingredient, only hyaluronic acid or a polyvinylpyrrolidone or apolyethyleneglycol stabilizer, the pharmaceutical composition retainsits potency substantially unchanged for six month, one year, two year,three year and/or four year periods when stored at a temperature betweenabout +8° C. and about −20° C. Additionally, the indicatedpharmaceutical compositions may have a potency or percent recovery ofbetween about 20% and about 100% upon reconstitution.

A pharmaceutical composition within the scope of the present inventionmay include a neurotoxin, and a hyaluronic acid. The hyaluronic acidstabilizes the neurotoxin. The pharmaceutical compositions disclosedherein may have a pH of between about 4 and 7.5 when reconstituted orupon injection. The hyaluronic acid in the instant pharmaceuticalcomposition is preferably combined with the instant botulinum toxin in aquantity of 0.1 to 10 mg, especially 1 mg hyaluronic acid per ml in a200 U/ml botulinum toxin solution. More preferably, the subject solutionalso contains a 1-100 mM, especially 10 mM sodium acetate buffer.

In another preferred embodiment, the composition may contain apolyalcohol as cryoprotectant. Examples of polyalcohols that might beused include, e.g., inositol, mannitol and other non-reducing alcohols.

It will be understood that the instant composition or preparation doesnot contain trehalose or maltotriose or related sugar or polyhydroxycompounds which are sometimes used as cryoprotectants.

The polyvinylpyrrolidone in the instant pharmaceutical composition ispreferably combined with the instant botulinum toxin in a quantity of 10to 500 mg, especially 100 mg polyvinylpyrrolidone per ml in a 200 U/mlbotulinum toxin solution. More preferably, the subject solution alsocontains a 1-100 mM, especially 10 mM sodium acetate buffer.

The polyethyleneglycol in the instant pharmaceutical composition ispreferably combined with the instant botulinum toxin in a quantity of 10to 500 mg, especially 100 mg polyethyleneglycol per ml in a 200 U/mlbotulinum toxin solution. More preferably, the subject solution alsocontains a 1-100 mM, especially 10 mM sodium acetate buffer.

Thus, the instant invention encompasses a botulinum toxin formulated ina pharmaceutical composition which contains a hyaluronic acid stabilizeror a polyvinylpyrrolidone stabilizer or a polyethyleneglycol stabilizer.Additionally, the pharmaceutical composition may contain a sodiumacetate buffer system and/or an alcoholic cryoprotectant. The followingexamples are provided by means of illustration only, and are notintended to be limiting.

The instant preparation or pharmaceutical composition is useful fortreating a condition for which botulinum neurotoxin therapy or treatmentis indicated. In one aspect it may be used for treating cosmeticconditions like wrinkling and pronounced wrinkling. In another aspect itmay be used for treating a condition where the condition is selectedfrom blepharospasm, hemifacial spasms, spasmodic torticollis,spasticities, migraine, low back pain, cervical spine disorders,strabismus, hyperhidrosis, hypersalivation, and dystonias. Furthermore,the instant preparation or composition is also used for themanufacturing of a medicament for a condition for which botulinumneurotoxin therapy is indicated, the condition being preferably selectedfrom cosmetic conditions, blepharospasm, hemifacial spasms, spasmodictorticollis, spasticities, migraine, low back pain, cervical spinedisorders, strabismus, hyperhidrosis, hypersalivation, and dystonias.Further medical indications treatable with the instant preparation orcomposition are, among others, benign cramping, essential tremor,mykomia, neurogenic muscle hypertrophy, palantal myoclonus, spinalmyoclonus, synkinesis/cranial nerve VII disorders, Duanne'seye-retraction syndrome, nystagmus, therapeutic ptosis for cornealprotection, oscillopsia, spasmodic dysphonia, granuloma, puberophonia,posterior glottic stenosis, rebalancing, stutter, TEP failure, essentialvoice tremor, vocal tics, cricopharyngeus, bruxism, masseterhypertrophy, morbid obesity, achalasia, anal fissure, anismus,intractable hiccups, severe constipation, anorectal pain, gastroparesis,benign anal disorders, esophageal diverticulosis, sphincter of Oddi,crocodile tears, sialocele, sialorrhea, drooling, parotid fistula,Frey's syndrome, ptyalism, detrusor-spincter dyssnergia, overactivebladder, vaginismus, urinary retention, hyperplasia, benignehyperplasia, tension headache, cervicogenic pain, myofascial pain,apraxia of eyelid opening, synkinesis secondary to facial nerve palsy,stuttering with glottal blocks, body odor, intrinsic rhinitis.

EXAMPLES

The botulinum toxin preparations of the instant invention, andpharmaceutical compositions thereof and method of treating therewith,are demonstrated to possess unique and advantageous properties,rendering the “subject matter as a whole”, as claimed herein unobvious.The botulinum toxin preparations and pharmaceutical compositions thereofhave exhibited, in standard accepted reliable test procedures, thefollowing valuable properties and characteristics:

Example 1 Botulinum Toxin Preparation

Pure neurotoxin from Clostridium botulinum type A is obtained by aprocess based on the process of DasGupta & Sathyamoorthy. Clostridiumbotulinum type A is cultivated in a 20 l fermenter in a mediumconsisting of 2% proteose peptone, 1% yeast extract, 1% glucose and0.05% sodium thioglycolate. After growth for 72 hours, the toxin isprecipitated by adding 3 N sulfuric acid (final pH=3.5). Theprecipitated and centrifuged biomass is extracted with 0.2 M sodiumphosphate buffer at pH 6.0.

After removal of the nucleic acids by precipitation with protaminesulfate, the toxin is precipitated by adding ammonium sulfate. Theprecipitate which has been solubilized and dialyzed against 50 mM sodiumphosphate at pH 6.0 is bound to a DEAE-Sephadex® column at the same pHand eluted with 150 mM NaCl. This is followed by a chromatography on aQAE-Sephadex® column which has been equilibrated with a 50 mM Tris/HClbuffer pH 7.9. The toxin is eluted via a NaCl gradient. In the laststep, the toxin is chromatographed on SP-Sephadex® at pH 7.0. In thiscase, the bound toxin is eluted from the column using a NaCl gradient(0-300 mM). The purified toxin is analyzed by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) and exhibits a purity of 95+/−5%. Thebiological activity is determined in the mouse LD50 assay: one LD50 unitcorresponds to 4.8 pg of protein.

Example 2 Finished Pharmaceutical Composition Containing Hyaluronic Acid

The purified neurotoxin of Example 1 was used to prepare a solutionwhich comprises 200 U botulinum toxin preparation and 1 mg hyaluronicacid per milliliter of distilled water. The solution was dispensed intovials.

Example 3 Finished Pharmaceutical Composition Containing Hyaluronic AcidAnd Sodium Acetate Buffer

The purified neurotoxin of Example 1 was used to prepare a solutionwhich comprises 200 U botulinum toxin preparation and 1 mg hyaluronicacid per milliliter of distilled water, and adjusted to a pH of 4.5, 5.0and 5.5 by addition of 10 mM sodium acetate buffer. The solution wasdispensed into vials.

Example 4 Determination of Botulinum Toxin Formulation Stability

The formulation of Example 2 was prepared and compared against botulinumtoxin formulated in Human Serum Albumin (HSA). On formulation, bothpreparations possessed the same activity. At 24 and 48 hours, thestability of the formulation of Example 2 matched that of the HSApreparation, with a loss of less than 5% of the initial activityoccurring in both samples.

Example 5 Determination of Botulinum Toxin Formulation Stability UnderVaried pH

The formulations of Example 3 were prepared and compared againstbotulinum toxin formulated in Human Serum Albumin (HSA). On formulation,both the instant preparations at every pH point and the HSA preparationpossessed the same activity. The pH 4.5 preparation exhibited a loss ofapproximately 50% of activity by the sixth day. The pH 5.0 and 5.5preparations lost all activity by the sixth day.

Example 6 Determination of Botulinum Toxin Formulation Stability UnderVaried pH and Lyophilization

The formulations of Example 3 were prepared and compared againstbotulinum toxin formulated in Human Serum Albumin (HSA). On formulation,the instant preparation at pH 4.5 possessed the same activity as the HSApreparation. More importantly, no loss of activity was detected onlyophilization.

Example 7 Finished Pharmaceutical Composition ContainingPolyvinylpyrrolidone

The purified neurotoxin of Example 1 was used to prepare a solutionwhich comprises 200 U botulinum toxin preparation and 100 mgpolyvinylpyrrolidone per milliliter of distilled water. The solution wasdispensed into vials.

Example 8 Finished Pharmaceutical Composition ContainingPolyvinylpyrrolidone and Sodium Acetate Buffer

The purified neurotoxin of Example 1 was used to prepare a solutionwhich comprises 200 U botulinum toxin preparation and 100 mgpolyvinylpyrrolidone per milliliter of distilled water, and adjusted toa pH of 4.5, 5.0 and 5.5 by addition of 10 mM sodium acetate buffer. Thesolution was dispensed into vials.

Example 8A Finished Pharmaceutical Composition ContainingPolyvinylpyrrolidone, Mannitol and Sodium Acetate Buffer

The purified neurotoxin of Example 1 was used to prepare a solutionwhich comprises 200 U botulinum toxin preparation, 100 mgpolyvinylpyrrolidone and 20 mg mannitol per milliliter of distilledwater, and adjusted to a pH of 4.5, 5.0 and 5.5 by addition of 10 mMsodium acetate buffer. The solution was dispensed into vials.

Example 8B Finished Pharmaceutical Composition ContainingPolyvinylpyrrolidone, Sorbitol and Sodium Acetate Buffer

The purified neurotoxin of Example 1 was used to prepare a solutionwhich comprises 200 U botulinum toxin preparation, 100 mgpolyvinylpyrrolidone and 20 mg sorbitol per milliliter of distilledwater, and adjusted to a pH of 4.5, 5.0 and 5.5 by addition of 10 mMsodium acetate buffer. The solution was dispensed into vials.

Example 9 Determination of Botulinum Toxin Formulation Stability

The formulation of Example 7 was prepared and compared against—botulinumtoxin formulated in Human Serum Albumin (HSA). On formulation, bothpreparations possessed the same activity.

Example 10 Determination of Botulinum Toxin Formulation Stability UnderVaried pH

The formulations of Example 8 were prepared and compared againstbotulinum toxin formulated in Human Serum Albumin (HSA). On formulation,both the instant preparations at every pH point and the HSA preparationpossessed the same activity. The pH 4.5 and 5.0 as well as the HSApreparations exhibited no loss of activity by within 24 hours offormulation. The pH 5.5 preparation lost 20% activity compared to theHSA and other preparations within 24 hours.

Example 10A Determination of Botulinum Toxin Formulation Stability UnderVaried pH

The formulations of Example 8A were prepared and compared againstbotulinum toxin formulated in Human Serum Albumin (HSA). On formulation,both the instant preparations at every pH point and the HSA preparationpossessed the same activity. The pH 4.5, 5.0 and 5.5 as well as the HSApreparations exhibited no loss of activity by within 24 hours offormulation.

Example 10B Determination of Botulinum Toxin Formulation Stability UnderVaried pH

The formulations of Example 8B were prepared and compared againstbotulinum toxin formulated in Human Serum Albumin (HSA). On formulation,both the instant preparations at every pH point and the HSA preparationpossessed the same activity. The pH 4.5, 5.0 and 5.5 as well as the HSApreparations exhibited no loss of activity within 24 hours offormulation.

Example 11 Determination of Botulinum Toxin Formulation Stability UnderVaried pH and Lyophilization

The formulations of Example 8 were prepared and compared againstbotulinum toxin formulated in Human Serum Albumin (HSA). On formulation,both the instant preparations at every pH point and the HSA preparationpossessed the same activity. More importantly, less than 10% loss ofactivity is detected on lyophilization for all formulations for up to 6months.

Example 11A Determination of Botulinum Toxin Formulation Stability UnderVaried pH and Lyophilization

The formulations of Example 8A were prepared and compared againstbotulinum toxin formulated in Human Serum Albumin (HSA). On formulation,both the instant preparations at every pH point and the HSA preparationpossessed the same activity. More importantly, less than 10% loss ofactivity is detected on lyophilization for all formulations for up to 6months.

Example 11B Determination of Botulinum Toxin Formulation Stability UnderVaried pH and Lyophilization

The formulations of Example 8B were prepared and compared againstbotulinum toxin formulated in Human Serum Albumin (HSA). On formulation,both the instant preparations at every pH point and the HSA preparationpossessed the same activity. More importantly, less than 10% loss ofactivity is detected on lyophilization for all formulations for up to 6months.

Example 12 Finished Pharmaceutical Composition ContainingPolyethyleneglycol

The purified neurotoxin of Example 1 was used to prepare a solutionwhich comprises 200 U botulinum toxin preparation and 100 mgpolyethyleneglycol per milliliter of distilled water. The solution wasdispensed into vials.

Example 12A Finished Pharmaceutical Composition ContainingPolyethyleneglycol and Mannitol

The purified neurotoxin of Example 1 was used to prepare a solutionwhich comprises 200 U botulinum toxin preparation, 100 mgpolyethyleneglycol and 20 mg mannitol per milliliter of distilled water.The solution was dispensed into vials.

Example 12B Finished Pharmaceutical Composition ContainingPolyethyleneglycol and Sorbitol

The purified neurotoxin of Example 1 was used to prepare a solutionwhich comprises 200 U botulinum toxin preparation, 100 mgpolyethyleneglycol and 20 mg sorbitol per milliliter of distilled water.The solution was dispensed into vials.

Example 13 Determination of Botulinum Toxin Formulation Stability

The formulation of Example 12 was prepared and compared againstbotulinum toxin formulated in Human Serum Albumin (HSA). On formulation,both preparations possessed the same activity.

Example 14 Determination of Botulinum Toxin Formulation Stability

The formulations of Example 12A and 12B were prepared and comparedagainst botulinum toxin formulated in Human Serum Albumin (HSA). Onformulation, both instant preparations and the HSA preparation possessedthe same activity. The instant preparations as well as the HSApreparation exhibited less than 20% loss of activity by within 24 hoursof formulation.

Example 15 Use of a Botulinum Toxin Pharmaceutical Composition

A 50 year old female seeks treatment for blepharospasm. Between about 10U and about 20 U of a botulinum toxin preparation of Example 3containing hyaluronic acid is injected intramuscularly into the patient.Within 1-7 days, the symptoms of blepharospasm are alleviated andalleviation of the symptoms persists for at least from about 2 months toabout 6 months.

Example 16 Use of a Botulinum Toxin Pharmaceutical Composition

A 50 year old female seeks treatment for blepharospasm. Between about 10U and about 20 U of a botulinum toxin preparation of Example 8containing polyvinylpyrrolidone is injected intramuscularly into thepatient. Within 1-7 days, the symptoms of blepharospasm are alleviatedand alleviation of the symptoms persists for at least from about 2months to about 6 months.

Example 17 Use of a Botulinum Toxin Pharmaceutical Composition

A 50 year old female seeks treatment for blepharospasm. Between about 10U and about 20 U of a botulinum toxin preparation of Example 12,containing polyethyleneglycol is injected intramuscularly into thepatient. Within 1-7 days, the symptoms of blepharospasm are alleviatedand alleviation of the symptoms persists for at least from about 2months to about 6 months.

Example 18 Use of a Botulinum Toxin Pharmaceutical Composition

A 50 year old female seeks treatment for blepharospasm. Between about 10U and about 20 U of a botulinum toxin preparation of Example 12A,containing polyethyleneglycol is injected intramuscularly into thepatient. Within 1-7 days, the symptoms of blepharospasm are alleviatedand alleviation of the symptoms persists for at least from about 2months to about 6 months.

Example 19 Use of a Botulinum Toxin Pharmaceutical Composition

A 50 year old female seeks treatment for blepharospasm. Between about 10U and about 20 U of a botulinum toxin preparation of Example 12B,containing polyethyleneglycol is injected intramuscularly into thepatient. Within 1-7 days, the symptoms of blepharospasm are alleviatedand alleviation of the symptoms persists for at least from about 2months to about 6 months.

1. A pharmaceutical composition consisting essentially of a botulinumneurotoxin from Clostridium botulinum of type A, B, C1, D, E, F or G ora mixture of two or more botulinum neurotoxins, wherein the neurotoxinor mixture of neurotoxins is free of the complexing proteins whichnaturally form complexes with botulinum neurotoxins, a non-proteinaceousstabilizing agent selected from hyaluronic acid, polyvinylpyrrolidoneand polyethyleneglycol, and a sodium acetate pH buffer, wherein thecomposition is freeze-dried.
 2. The pharmaceutical composition of claim1, wherein the botulinum neurotoxin is modified chemically or modifiedby genetic manipulation, or is a mixture thereof.
 3. The pharmaceuticalcomposition of claim 1, wherein the non-proteinaceous stabilizing agentis hyaluronic acid.
 4. The pharmaceutical composition of claim 1,wherein the pH of the composition prior to freeze-drying is in a rangeof about 4.5 to about 5.5.
 5. The pharmaceutical composition of claim 1,wherein the pH of the composition after reconstitution is a range ofabout 6.5 to about 7.5.
 6. The pharmaceutical composition of claim 1,for administration to an animal, including a human, in an amounteffective for the treatment of a condition for which botulinumneurotoxin therapy or treatment is indicated.
 7. The pharmaceuticalcomposition of claim 6, wherein the condition for which botulinumneurotoxin therapy or treatment is indicated is selected from cosmeticconditions, blepharospasm, hemifacial spasms, spasmodic torticollis,spasticities, dystonias, migraine, low back pain, cervical spinedisorders, strabismus, hyperhidrosis and hypersalivation.
 8. Thepharmaceutical composition of claim 7, wherein the cosmetic condition ispronounced wrinkling.